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Antimicrobial Agents and Chemotherapy, January 2000, p. 10-13, Vol. 44, No. 1
Center for Adaptation Genetics and Drug
Resistance1 and the Departments of
Molecular Biology and Microbiology2 and
of Medicine,5 Tufts University School of
Medicine, Boston, Massachusetts 02111, Herz-und Diabeteszentrum
NRW, Bad Oeynhausen,3 and Section of
Infectious Diseases and Clinical Immunology, University Hospital and
Medical Center, Ulm,4 Germany
Received 1 July 1999/Returned for modification 31 August
1999/Accepted 29 September 1999
Fluoroquinolone-resistant mutants, selected from a wild-type
Escherichia coli K-12 strain and its Mar mutant by exposure
to increasing levels of ofloxacin on solid medium, were analyzed by
Northern (RNA) blot analysis, sequencing, and radiolabelled ciprofloxacin accumulation studies. Mutations in the target gene gyrA (DNA gyrase), the regulatory gene marR,
and additional, as yet unidentified genes (genes that probably affect
efflux mediated by the multidrug efflux pump AcrAB) all contributed to
fluoroquinolone resistance. Inactivation of the acrAB locus
made all strains, including those with target gene mutations,
hypersusceptible to fluoroquinolones and certain other unrelated drugs.
These studies indicate that, in the absence of the AcrAB pump, gyrase
mutations fail to produce clinically relevant levels of fluoroquinolone resistance.
Fluoroquinolone (FQ) resistance in
Escherichia coli can be caused by mutations in the target
topoisomerases of the drugs, DNA gyrase (e.g., in gyrA)
(11, 30) and topoisomerase IV (e.g., in parC)
(10, 13, 14). Mutations that affect regulatory genes such as
marA (3, 5) or soxS (2)
also lead to FQ resistance. The latter genes regulate intracellular
drug concentrations by producing decreased uptake and/or increased
efflux of the drug (1). In E. coli,
overexpression of MarA causes decreased expression of the OmpF porin
(6) as well as increased expression of the multidrug efflux
pump AcrAB (23), thereby conferring resistance to a large
number of antimicrobial agents (for a review, see reference 1). Studies with mutants selected in vitro have been
confined to descriptions of phenotypic differences (MICs, outer
membrane profiles, accumulation of FQs) or have focused on mutations in the quinolone resistance-determining regions (QRDRs) of gyrA
and/or parC (10, 25, 27, 29). In this in vitro
study we sought to understand the role of the AcrAB efflux pump in the
FQ resistance mediated by topoisomerase mutations acquired during
selection on ofloxacin. The AcrAB efflux pump was found to be critical
to the FQ resistance level.
(This study was presented in part at the 38th Interscience Conference
on Antimicrobial Agents and Chemotherapy, 24 to 27 September 1998, San
Diego, Calif.)
Antibiotics, chemicals, and media.
Ofloxacin (OFL) was
kindly donated by Hoechst, Frankfurt, Germany. Radiolabelled
[14C]ciprofloxacin ([14C]CIP) was a
generous gift of Bayer AG, Leverkusen, Germany. Carbonyl cyanide
m-chlorophenylhydrazone (CCCP) was purchased from Sigma Chemical Co., St. Louis, Mo., and organic solvents were purchased from
Aldrich, Milwaukee, Wis. Strains were grown in Luria-Bertani (LB) broth
(10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl per
liter) unless otherwise noted.
Bacterial strains and plasmids.
All strains were derivatives
of plasmid-free E. coli K-12 strain AG100 (8) and
its Mar mutant AG112. The latter was selected on tetracycline in two
steps (this study). Wild-type E. coli GC4468, its derived
Sox mutant JTG1078 (soxR105 [9]), and
plasmid pSXS bearing the soxS gene on a 432-bp fragment
(2) were a generous gift of B. Demple. Strain JZM120
(JC7623 Selection of FQ-resistant mutants.
Mutants 1-AG100, 2-AG100,
3*-AG100, and 4*-AG100 and mutants 1-AG112, 2-AG112, and 3-AG112 were
sequential step mutants derived from strains AG100 and AG112,
respectively, on solid media. For each step, about 1011
cells of an overnight culture were plated on several LB agar plates
supplemented with increasing concentrations of OFL (0.5 to 16 µg/ml).
Single colonies were further purified on OFL-supplemented agar plates.
Mutants 1-AG100 and 2-AG100 came out of one series, while mutants
3*-AG100 and 4*-AG100 came from a second series.
Susceptibility testing.
The MICs of selected antimicrobial
agents were determined by a standard broth microdilution procedure with
cation-adjusted Mueller-Hinton broth (Becton Dickinson, Cockeysville,
Md.) and an inoculum of 5 × 105 CFU/ml according to
National Committee for Clinical Laboratory Standards (NCCLS)
performance and interpretive guidelines (20). The
antimicrobial agents in the commercially available microtiter plates
(Merlin Diagnostics GmbH, Bornheim, Germany) included the FQs CIP,
enoxacin, fleroxacin, norfloxacin, OFL, pefloxacin, sparfloxacin (SPX),
and trovafloxacin (TVA). They also included tetracycline (TET),
chloramphenicol (CML), trimethoprim, cefoxitin (CFOX), cefaclor,
cefixime, and loracarbef. The MICs of bile salts were determined by a
broth macrodilution procedure: sodium cholate and sodium deoxycholate
were serially diluted in twofold steps in LB broth, and tubes were
inoculated with 5 × 105 CFU/ml. The MICs of both bile
salts (data not shown) and antibiotics were determined twice in
independent experiments, with reproducible results.
P1 transduction.
acrAB-deleted strains were
constructed by P1 transduction (26) of
DNA sequencing.
The QRDRs of gyrA (nucleotides
123 to 366) or parC (nucleotides 145 to 492) in the
FQ-resistant mutants were amplified by PCR and were purified by use of
Qiaquick spin columns (Qiagen, Hilden, Germany), as described
previously (7; S. Conrad, L. Scheit, M. Oethinger,
G. Klotz, R. Marre, and W. V. Kern, Abstr. 36th Intersci. Conf.
Antimicrob. Agents Chemother, abstr. C9, 1996). marOR (which
encodes the operator [marO] and repressor [marR] of the Mar operon) was amplified from base pairs
1311 to 1858 with primer pair ORAB2 and RK3 as described earlier
(22). Direct cycle sequencing in both directions was
performed with the same primers in an automatic 373A DNA Sequencer
(Applied Biosystems).
RNA extraction and Northern blot analysis.
Northern blot
analysis was performed as described previously (22). In
brief, RNA was harvested by a cesium chloride method from
mid-logarithmic-phase cultures grown at 30°C. For assessment of the
state of the marRAB operon or the soxRS operon,
cultures were split and half the cultures were induced with 5 mM sodium salicylate (marRAB operon) or 1.3 mM paraquat
(soxRS operon). The level of transcription from both operons
was assessed by hybridization of radiolabelled DNA probes
(marA or soxS) to the membrane-bound RNA (20 µg/lane), exposure on a PhosphoImager screen, and visualization with
Image-Quant Software (Molecular Dynamics, Sunnyvale, Calif.), as
described recently (22).
Accumulation of [14C]CIP in whole cells.
Cultures were grown to the logarithmic phase in LB broth at 30°C,
washed in 50 mM potassium phosphate-0.2% glucose (pH 7.4), and
resuspended in the same buffer to an optical density at 600 nm
(OD600) of 5 to 7. [14C]CIP (specific
activity, 59 mCi/mmol) was added to 10 µM. Accumulation was measured
at equilibrium after 5 and 15 min at 30°C by dilution of 50 µl of
the cell-labelling suspension into 5 ml of 100 mM LiCl-50 mM
KPO4 (pH 7.4), collection of cells immediately on Metricel mixed-cellulose ester membrane filters (pore size, 0.45 µm; Gelman Sciences Inc., Ann Arbor, Mich.), and washing with 5 ml of the same
buffer. The filters were dried, and the radioactivity was assayed with
a liquid scintillation counter by using Betafluor (National
Diagnostics, Somerville, N.J.). The counting efficiency was 90%. The
amount of radiolabel that bound to filters in the absence of cells was
subtracted. When used, CCCP, which destroys the proton motive force,
was added at 20 min to a final concentration of 200 µM, and the
levels of accumulation of CIP were assayed 5 and 15 min thereafter. The
assay was designed in a way to investigate up to five strains in one
experiment and to include, in addition, AG112 as a control strain.
Results were calculated as accumulation of CIP in picomoles per
OD600 unit, where 1 OD600 unit represented the
number of cells in 1 ml when the OD600 was equal to 1 (approximately 109 E. coli cells; about 0.3 mg
of protein). The ratio (expressed as percent) of CIP accumulation of
energized cells divided by that of deenergized (CCCP-treated) cells was
used as an indirect measure of active efflux (15). The
values used to obtain this ratio were the separate averages of
accumulation before (5 and 15 min) and after (25 min and 35 min) the
addition of CCCP.
Selection of FQ-resistant mutants in vivo.
The number of
mutants observed for the total number of cells plated at the different
levels of OFL ranged between 8 × 10
0066-4804/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Ineffectiveness of Topoisomerase Mutations in
Mediating Clinically Significant Fluoroquinolone Resistance in
Escherichia coli in the Absence of the AcrAB Efflux
Pump
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
acrAB::Tn903
Kanr) (17) was kindly supplied by H. Nikaido.
acrAB::Tn903 Kanr from
strain JZM120 into AG100, AG112, and all derived FQ-resistant mutants
(18). The AcrAB gene-deleted strains were designated with
the suffix "AK", e.g., AG100AK, 1-AG100AK, and AG112AK. Two independent transductants were saved for each recipient. Deletion of
the gene for AcrAB was confirmed by the absence of intact target DNA in
a PCR assay and by greatly increased susceptibility to bile salts (data
not shown), as reported previously (29). Besides positive
and negative controls in each reaction, the gapA gene was
used as the internal standard for determination of the validities of
the PCR assays.
RESULTS AND DISCUSSION
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
8 and 1 × 1010, which agrees well with previous data (10, 27,
29). None of the mutants was defective in growth. The first
mutation step increased resistance to OFL by eightfold in both AG100
and its Mar mutant AG112 (Table 1).
Subsequent increases were two- to fourfold in all steps (Table 1 and
Table 2), with the highest MIC being that
of OFL for 3-AG112 (MIC, 8 µg/ml). The MICs of all FQs increased in
parallel, with the order of MICs being OFL > CIP > TVA = SPX (Table 2).
TABLE 1.
FQ resistance mutations in E. coli AG100 and
its Mar mutant AG112a
TABLE 2.
Effect of deletion of acrAB on susceptibility
of FQ-resistant mutants of E. coli AG100
and AG112a
Identification of chromosomal mutations in structural and regulatory genes and susceptibilities to unrelated antibiotics. Sequencing of the QRDRs of gyrA revealed that an identical point mutation at codon 87 (substitution of glycine for aspartate) occurred during the first mutation step in both AG100- and AG112-derived mutants (Table 1). These mutations led to an increase in the MICs of FQs without an additional multiple resistance (Mar) phenotype. There are several reports that a gyrA mutation is the first "visible" mutation in the chain of events that leads to higher levels of FQ resistance during stepwise in vitro mutagenesis (10, 25; W. V. Kern, M. Oethinger, A. S. Ritter, S. Conrad, R. Marre, and S. B. Levy, Abstr. 37th Intersci. Conf. Antimicrob. Agents Chemother., abstr. C-182, 1997). That the first gyrA mutations in both AG100 and AG112 were identical may be coincidental.
The second-step mutation in the AG100 background was a Mar mutation, as shown by overexpression of marRAB by Northern blot analysis of 2-AG100. Such overexpression was found in three independently selected second-step mutants of AG100 (data not shown). Constitutive overexpression of marA was associated with increased levels of resistance to TET, CML, and CFOX (Table 2). Sequencing of marOR of a third-step mutant, mutant 3*-AG100, from a different series showed a different mar mutation but the same gyrA mutation. As expected (9), marRAB was derepressed in Mar mutant AG112 and all subsequently derived mutants. Sequencing of marOR of the parental strain AG112 identified a 5-bp deletion after the codon for amino acid 12 of MarR, resulting in deletion of one amino acid and a change in the complete protein sequence thereafter (Table 1). A single mutation in the gyrA gene conferred a somewhat higher level of FQ resistance than overexpression of marA by itself did: the OFL MICs were 0.25 µg/ml (eightfold increase) and 0.125 µg/ml (fourfold increase) for 1-AG100 and AG112, respectively (Table 1). The two mutations are multiplicative (OFL MICs, 1 µg/ml [32-fold increase] for 2-AG100 and 1-AG112; Table 1). During further steps to higher levels of FQ resistance, a mutant in another series, mutant 4*-AG100, acquired a second mutation in gyrA, at codon 83, substituting leucine for serine (Table 1). No additional mutations in gyrA, parC, or marOR could be identified in any of the more resistant mutants, nor did they constitutively overexpress soxS at any step (data not shown). Mutant 2-AG112, derived from gyrA marR double mutant 1-AG112, displayed increased levels of resistance to multiple drugs (Table 2), with no further mutation in marOR. The additional mutation possibly led to upregulation of acrAB (see below). In contrast, the next step mutant, 3-AG112, had increased levels of resistance to only FQs and CFOX (Table 2). The genetic basis for this resistance is also unknown.Accumulation of [14C]CIP in whole cells. The accumulation of CIP in whole energized cells reached a plateau by 5 min and averaged 103 pmol of CIP/OD600 unit in AG100. When the proton motive force was dissipated by adding 200 µM CCCP, the level of accumulation of [14C]ciprofloxacin doubled (201 pmol of CIP/OD600 unit). These findings confirmed earlier studies with norfloxacin (4) that FQ-susceptible E. coli cells use energy to reduce the level of FQ accumulation; i.e., they show active efflux. This phenomenon was also observed for Proteus vulgaris with OFL (12). In comparison, accumulation in AG112 was 54% of that in the wild-type strain, averaging 56 pmol of CIP/OD600 unit, and increased to 226 pmol CIP/OD600 unit after the addition of CCCP. The rapid accumulation of CIP (this work) or norfloxacin (4) to the level in the AG100 parental strains upon deenergization of the cells rules out downregulation of outer membrane porins as the major mechanism of reduced levels of drug accumulation in Mar mutants. The amount of CIP accumulated by energized first-step (gyrA) mutants 1-AG100 and 1-AG112 and the increase in the level of drug uptake following deenergization were virtually identical to those for parental strains AG100 and AG112, respectively (data not shown). In second-step Mar mutant 2-AG100, the level of accumulation decreased to 65 pmol CIP/OD600 unit (63% of that for the wild-type strain; Fig. 1). Independently isolated mutants 3*-AG100 and 4*-AG100 showed even lower levels of accumulation (16 and 19% of that for the wild-type strain, respectively; Fig. 1). Similarly, mutants 2-AG112 and 3-AG112 accumulated considerably less CIP than Mar mutants AG112 and 1-AG112 (Fig. 1). With less drug accumulation, the cells can survive in the presence of higher external concentrations of FQ. The mutations also increased the cells' resistance to TET, CML, and CFOX.
|
Effects of deletion of acrAB.
Upon deletion of the AcrAB
multidrug efflux pump, the ability to actively efflux CIP was
completely lost by all strains (data not shown). Energized AG100 cells
bearing the acrAB deletion accumulated CIP to more than
twice the level seen for energized acrAB-positive cells
(Fig. 1). No difference in the levels of CIP uptake was noted between
any acrAB-deleted strains, irrespective of their mutations
in marRAB or gyrA (Fig. 1). Although all
acrAB strains became profoundly hypersusceptible to
all FQs, mutants with newly acquired mutations in gyrA
(Table 1) still retained some gyrA-mediated FQ resistance
(Table 2), although this was therefore well below the level of clinical
significance. The OFL MIC for the gyrA double mutant
4*-AG100 acrAB, for instance, was only 0.125 µg/ml.
Interestingly, the differences in MICs of different FQs were no longer
observed for acrAB strains, e.g., for 2-AG100
acrAB, the OFL, CIP, and TVA MICs were 0.06 µg/ml,
while for acrAB-positive strain 2-AG100, the OFL MIC was 1 µg/ml, the CIP MIC was 0.5 µg/ml, and the TVA MIC was 0.25 µg/ml
(Table 2). The SPX MICs for all acrAB mutants were below
the detection limit (MICs, 
0.015 µg/ml). Deletion of
acrAB also eliminated the multidrug resistance conferred by overexpression of marA, thus underlining previous findings
that the AcrAB multidrug efflux pump plays a major role in the
antibiotic resistance phenotype of Mar mutants (23). Effects
of additional, as yet unknown mutations were also completely abolished
by deletion of acrAB (Table 2). We conclude, therefore, that
the effect of one or more additional mutations on drug resistance is
also affected by acrAB.
Concluding remarks. In view of the large number of pumps in E. coli (21, 24), it is remarkable that none of the other pumps actively effluxes CIP in the absence of the AcrAB efflux pump. Thus, the AcrAB multidrug efflux pump appears to be the only, or at least the most important, pump in E. coli which uses CIP as a substrate.
Our findings correspond with recent data on the triclosan susceptibility of E. coli, which was greatly affected by loss of the AcrAB pump (18). While overexpression of acrAB, marA, or soxS increased the cells' resistance to triclosan about twofold, a mutation in the target of triclosan, enoyl reductase (encoded by fabI), rendered the cell about 100-fold more resistant (19). However, deletion of acrAB reduced the level of resistance 10-fold in all strains, rendering the fabI mutation less effective, similar to the decrease in the effectiveness of topoisomerase mutations in the case of FQs. The prominent finding of our work is that the AcrAB efflux pump has a powerful role in both the intrinsic and the acquired level of FQ resistance in E. coli. As previously reported by others for Pseudomonas (21), we show that efflux mechanisms significantly decrease the action of FQs also in E. coli, even though in the latter organism the hydrophilic drugs presumably traverse the outer membrane more rapidly through the more permeable porin channels (21). Mutations in unidentified chromosomal loci, not marOR or soxRS, modulate the level of resistance apparently by increasing efflux via AcrAB. Blockage of the AcrAB efflux pump would increase the potencies of drugs such as FQs even in the face of topoisomerase mutations. While this work was in progress, a report on the importance of the Mex multidrug efflux pumps for FQ resistance in Pseudomonas aeruginosa drew a parallel conclusion (16).| |
ACKNOWLEDGMENTS |
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This study was supported in part by a research grant from the U.S. Public Health Service (grant GM 51661 to S.B.L. and L.M.M.), the Deutsche Forschungsgemeinschaft (grant Oe 195/1-1 to M.O.), and the University of Ulm (grants Ke700/1-1 and P172/1994 to M.O. and W.V.K.).
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FOOTNOTES |
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* Corresponding author. Mailing address: Center for Adaptation Genetics and Drug Resistance, Tufts University School of Medicine, 136 Harrison Ave., Boston, MA 02111. Phone: (617) 636-6764. Fax: (617) 636-0458. E-mail: slevy{at}opal.tufts.edu.
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Abstract
Introduction
Materials and Methods
Results and Discussion
References
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Fange, D., Nilsson, K., Tenson, T., Ehrenberg, M.
(2009). Drug efflux pump deficiency and drug target resistance masking in growing bacteria. Proc. Natl. Acad. Sci. USA
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Preisler, A., Heisig, P.
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Morgan-Linnell, S. K., Becnel Boyd, L., Steffen, D., Zechiedrich, L.
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Webber, M. A., Randall, L. P., Cooles, S., Woodward, M. J., Piddock, L. J. V.
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Bohnert, J. A., Schuster, S., Fahnrich, E., Trittler, R., Kern, W. V.
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59: 1216-1222
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Nicoloff, H., Perreten, V., Levy, S. B.
(2007). Increased Genome Instability in Escherichia coli lon Mutants: Relation to Emergence of Multiple-Antibiotic-Resistant (Mar) Mutants Caused by Insertion Sequence Elements and Large Tandem Genomic Amplifications. Antimicrob. Agents Chemother.
51: 1293-1303
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Coldham, N. G., Randall, L. P., Piddock, L. J. V., Woodward, M. J.
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(2006). MarA-Like Regulator of Multidrug Resistance in Yersinia pestis.. Antimicrob. Agents Chemother.
50: 2971-2975
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Saito, R., Sato, K., Kumita, W., Inami, N., Nishiyama, H., Okamura, N., Moriya, K., Koike, K.
(2006). Role of type II topoisomerase mutations and AcrAB efflux pump in fluoroquinolone-resistant clinical isolates of Proteus mirabilis. J Antimicrob Chemother
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Nicoloff, H., Perreten, V., McMurry, L. M., Levy, S. B.
(2006). Role for Tandem Duplication and Lon Protease in AcrAB-TolC- Dependent Multiple Antibiotic Resistance (Mar) in an Escherichia coli Mutant without Mutations in marRAB or acrRAB.. J. Bacteriol.
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Ricci, V., Tzakas, P., Buckley, A., Coldham, N. C., Piddock, L. J. V.
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Ge, B., McDermott, P. F., White, D. G., Meng, J.
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Pumbwe, L., Randall, L. P., Woodward, M. J., Piddock, L. J. V.
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Burgos, J. M., Ellington, B. A., Varela, M. F.
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Tavio, M. M, Vila, J., Perilli, M., Casanas, L. T, Macia, L., Amicosante, G., Jimenez de Anta, M. T
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Baucheron, S., Tyler, S., Boyd, D., Mulvey, M. R., Chaslus-Dancla, E., Cloeckaert, A.
(2004). AcrAB-TolC Directs Efflux-Mediated Multidrug Resistance in Salmonella enterica Serovar Typhimurium DT104. Antimicrob. Agents Chemother.
48: 3729-3735
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Yu, X., Susa, M., Knabbe, C., Schmid, R. D., Bachmann, T. T.
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Peric, M., Bozdogan, B., Galderisi, C., Krissinger, D., Rager, T., Appelbaum, P. C.
(2004). Inability of L22 ribosomal protein alteration to increase macrolide MICs in the absence of efflux mechanism in Haemophilus influenzae HMC-S. J Antimicrob Chemother
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Linde, H.-J., Lehn, N.
(2004). Mutant prevention concentration of nalidixic acid, ciprofloxacin, clinafloxacin, levofloxacin, norfloxacin, ofloxacin, sparfloxacin or trovafloxacin for Escherichia coli under different growth conditions. J Antimicrob Chemother
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Miller, K., O'Neill, A. J., Chopra, I.
(2004). Escherichia coli Mutators Present an Enhanced Risk for Emergence of Antibiotic Resistance during Urinary Tract Infections. Antimicrob. Agents Chemother.
48: 23-29
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Schneiders, T., Amyes, S. G. B., Levy, S. B.
(2003). Role of AcrR and RamA in Fluoroquinolone Resistance in Clinical Klebsiella pneumoniae Isolates from Singapore. Antimicrob. Agents Chemother.
47: 2831-2837
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Gruteke, P., Goessens, W., van Gils, J., Peerbooms, P., Lemmens-den Toom, N., van Santen-Verheuvel, M., van Belkum, A., Verbrugh, H.
(2003). Patterns of Resistance Associated with Integrons, the Extended-Spectrum {beta}-Lactamase SHV-5 Gene, and a Multidrug Efflux Pump of Klebsiella pneumoniae Causing a Nosocomial Outbreak. J. Clin. Microbiol.
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Mc Dermott, P. F., Walker, R. D., White, D. G.
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Bina, X., Perreten, V., Levy, S. B.
(2003). The Periplasmic Protein MppA Requires an Additional Mutated Locus To Repress marA Expression in Escherichia coli. J. Bacteriol.
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Webber, M. A., Piddock, L. J. V.
(2003). The importance of efflux pumps in bacterial antibiotic resistance. J Antimicrob Chemother
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Baranwal, S., Dey, K., Ramamurthy, T., Nair, G. B., Kundu, M.
(2002). Role of Active Efflux in Association with Target Gene Mutations in Fluoroquinolone Resistance in Clinical Isolates of Vibrio cholerae. Antimicrob. Agents Chemother.
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Lin, J., Michel, L. O., Zhang, Q.
(2002). CmeABC Functions as a Multidrug Efflux System in Campylobacter jejuni. Antimicrob. Agents Chemother.
46: 2124-2131
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Chollet, R., Bollet, C., Chevalier, J., Mallea, M., Pages, J.-M., Davin-Regli, A.
(2002). mar Operon Involved in Multidrug Resistance of Enterobacter aerogenes. Antimicrob. Agents Chemother.
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Notka, F., Linde, H.-J., Dankesreiter, A., Niller, H.-H., Lehn, N.
(2002). A C-terminal 18 amino acid deletion in MarR in a clinical isolate of Escherichia coli reduces MarR binding properties and increases the MIC of ciprofloxacin. J Antimicrob Chemother
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RANDALL, L. P., COOLES, S. W., SAYERS, A. R., WOODWARD, M. J.
(2001). Association between cyclohexane resistance in Salmonella of different serovars and increased resistance to multiple antibiotics, disinfectants and dyes. J Med Microbiol
50: 919-924
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Mao, W., Warren, M. S., Lee, A., Mistry, A., Lomovskaya, O.
(2001). MexXY-OprM Efflux Pump Is Required for Antagonism of Aminoglycosides by Divalent Cations in Pseudomonas aeruginosa. Antimicrob. Agents Chemother.
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Chopra, I., Roberts, M.
(2001). Tetracycline Antibiotics: Mode of Action, Applications, Molecular Biology, and Epidemiology of Bacterial Resistance. Microbiol. Mol. Biol. Rev.
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Jellen-Ritter, A. S., Kern, W. V.
(2001). Enhanced Expression of the Multidrug Efflux Pumps AcrAB and AcrEF Associated with Insertion Element Transposition in Escherichia coli Mutants Selected with a Fluoroquinolone. Antimicrob. Agents Chemother.
45: 1467-1472
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Wang, H., Dzink-Fox, J. L., Chen, M., Levy, S. B.
(2001). Genetic Characterization of Highly Fluoroquinolone-Resistant Clinical Escherichia coli Strains from China: Role of acrR Mutations. Antimicrob. Agents Chemother.
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Giraud, E., Leroy-Setrin, S., Flaujac, G., Cloeckaert, A., Dho-Moulin, M., Chaslus-Dancla, E.
(2001). Characterization of high-level fluoroquinolone resistance in Escherichia coli O78:K80 isolated from turkeys. J Antimicrob Chemother
47: 341-343
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Lomovskaya, O., Warren, M. S., Lee, A., Galazzo, J., Fronko, R., Lee, M., Blais, J., Cho, D., Chamberland, S., Renau, T., Leger, R., Hecker, S., Watkins, W., Hoshino, K., Ishida, H., Lee, V. J.
(2001). Identification and Characterization of Inhibitors of Multidrug Resistance Efflux Pumps in Pseudomonas aeruginosa: Novel Agents for Combination Therapy. Antimicrob. Agents Chemother.
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Mazzariol, A., Tokue, Y., Kanegawa, T. M., Cornaglia, G., Nikaido, H.
(2000). High-Level Fluoroquinolone-Resistant Clinical Isolates of Escherichia coli Overproduce Multidrug Efflux Protein AcrA. Antimicrob. Agents Chemother.
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Piddock, L. J. V., White, D. G., Gensberg, K., Pumbwe, L., Griggs, D. J.
(2000). Evidence for an Efflux Pump Mediating Multiple Antibiotic Resistance in Salmonella enterica Serovar Typhimurium. Antimicrob. Agents Chemother.
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Poole, K.
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Linde, H.-J., Notka, F., Metz, M., Kochanowski, B., Heisig, P., Lehn, N.
(2000). In Vivo Increase in Resistance to Ciprofloxacin in Escherichia coli Associated with Deletion of the C-Terminal Part of MarR. Antimicrob. Agents Chemother.
44: 1865-1868
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Lee, A., Mao, W., Warren, M. S., Mistry, A., Hoshino, K., Okumura, R., Ishida, H., Lomovskaya, O.
(2000). Interplay between Efflux Pumps May Provide Either Additive or Multiplicative Effects on Drug Resistance. J. Bacteriol.
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